DCs and TEVs were isolated as previously described. DCs were plated on Labtek II chambers with chamber protectors coated with fibronectin (5 μg/mL) and differentiated in the same manner as previously described. TEVs from MC38 cell lines were stained with DiO lipophilic dye (Invitrogen) for 30 min and washed three times with PBS and ultracentrifugation at 100,000 × g for 70 min at 4°C. TEV pellets labeled with DiO were suspended in DC medium, and 1 μg TEVs were added to DC cultures on day 6 with 100 ng/mL LPS. Following 24 h to allow for TEV uptake, DC culture on glass slides was stained with Cytopainter Red (Abcam, cat. no. ab219942) for 30 min according to the manufacturer’s instructions and fixed with 4% formaldehyde for 30 min and washed 5 times with PBS. Chamber protectors were removed, slides were mounted with mounting medium with DAPI, and DCs exposed to MC38 TEVs were imaged on the BZX810 fluorescence microscope (Keyence). A series of photos were taken with a (4.4 μm) stepwise increase (0.4 μm) at each step on the z axis to validate the intracellular uptake of TEVs prior to the in vivo experiment.
Dio lipophilic dye
Manufactured by Thermo Fisher Scientific
DiO is a lipophilic dye that can be used for staining cell membranes. It has green fluorescence when excited at appropriate wavelengths.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Cytopainter red, by Abcam (2 mentions)
Bz x810 fluorescence microscope, by Keyence (2 mentions)
Cellquest 3, by BD (2 mentions)
Diva software, by BD (2 mentions)
A1 32 edge 5mm 20 177 a32, by NeuroNexus (2 mentions)
Rhd2000 usb interface board, by Intan Technologies (2 mentions)
Interleukin 2 (il 2), by Immunotools (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Facs fortessa, by BD (1 mentions)
8 protocols using dio lipophilic dye
1
Intracellular Uptake of Tumor-Derived Extracellular Vesicles by Dendritic Cells
2
Visualizing Tumor-Derived EV Uptake by DCs
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DCs and TEVs were isolated as previously described. DCs were plated on Labtek II chambers with chamber protectors coated with fibronectin (5ug/mL) and differentiated in the same manner as previously described. TEVs from MC38 cell lines were stained with DiO lipophilic dye (Invitrogen) for 30 minutes and washed three times with PBS and ultracentrifugation at 100,000g for 70 minutes at 4 C. TEV pellets labelled with DiO were suspended in DC media, and 1μg TEVs were added to DC cultures on Day 6 with 100ng/mL LPS. Following 24 hours to allow for TEV uptake DCs culture on glass slides was stained with Cytopainter Red (cat# ab219942, Abcam) for 30 minutes according to the manufactures instructions and fixed with 4% formaldehyde for 30 minutes and washed 5 times with PBS. Chamber protectors were removed, slides were mounted with mounting media with DAPI, and DCs exposed to MC38 TEVs were imaged on the BZX810 fluorescence microscope (Keyence). A series of photos were taken with a (4.4 μm) stepwise increase (0.4 μm) at each step on the z-axis to validate the intracellular uptake of TEVs prior to the in vivo experiment.
3
Evaluating DC/shIL-10R/TAg Activation of Primary Antigen
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To evaluate the ability of DC/shIL-10R/TAg to activate the primary antigen For this purpose, the 5-days co-culture of mature DCs (0.18×106 cells) (i.e. stimulated with MC38-derived TAg) and naive spleen cells (splenocytes) (1.8×106 cells) in the presence of recombinant human (rh) IL−2 (200 U/ml, Immunotools) was performed. After this time, cells were harvested and the cytotoxic activity of effector splenocytes against target (MC38) cells stained with DiO lipophilic dye (Molecular Probes) was analyzed according to a previously described procedure (42 (link)). Two E:T (effector to target) ratios were investigated: 10:1 and 30:1. The dead target cells were distinguished with propidium iodide (PI) solution and the percentage of DiO+PI+ MC38 cells was determined using a FACS Calibur with CellQuest 3.3 software (Becton Dickinson).
4
Evaluating Cytotoxic Activity of Effector Splenocytes
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Spleen single-cell suspensions (2×106 cells) were thawed and cocultured with mitomycin C-treated MC38 cells (0.1×106 cells) in the presence of rhIL−2 (200 U/ml). After 5 days of restimulation, cells were harvested and the cytotoxic activity of effector splenocytes against target (MC38) cells stained with DiO lipophilic dye (Molecular Probes) was analyzed according to the previously described procedure (38 (link)). Two E:T (effector to target) ratios were investigated: 10:1 and 30:1. The dead target cells were distinguished with propidium iodide (PI, Life Technologies) solution and the percentage of DiO+PI+ MC38 cells was determined. The degranulation assay was used as described earlier (section: Primary stimulation of splenic cells by transduced dendritic cells). Supernatants were collected and stored at 4°C until ELISA was performed.
5
Cytotoxic Activity Analysis of Tumor-Specific T Cells
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Spleen cells obtained from control or treated tumor-bearing mice were cocultured with mitomycin C-treated MC38 cells (50 mg MitC/3 × 106 cells; 30 min., 37°C) in the presence of recombinant human IL-2 (200 U/ml). After 5 days of restimulation, supernatants were collected and stored in 4°C. Cytotoxic activity of cells with application of DiO lipophilic dye (Molecular Probes) were analyzed according to previously described procedure (28 (link)). The percentage of dead double positive (DiO+PI+) MC38 cells was determined after analysis using FACS Calibur with CellQuest software (Becton Dickinson). In order to determine the percentage of CD107+ cells, restimulated spleen cells were incubated for 2 h with MC38 cells in the presence of monoclonal anti-CD107 antibody conjugated with APC (BioLegend). After that, cells were stained with anti-CD8 PE-Cy7 and anti-CD49b PE and analyzed using LSR Fortessa with Diva software (Becton Dickinson).
6
Evaluating Cytotoxic T Cell Activity
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Spleen cells obtained from non-treated or treated tumor-bearing mice were cocultured with mitomycin C-treated MC38 cells (50 mg mitomycin C/3×106 cells, 30 min., 37°C) in the presence of recombinant human IL-2 (200 U/ml). After 5 days of restimulation, supernatants were collected and stored at 4°C until ELISA was performed. Cytotoxic activity of cells stained with DiO lipophilic dye (Molecular Probes) was analyzed according to a previously described procedure (47 (link)). Two E:T (effector to target) ratios were investigated: 10:1 and 30:1. The percentage of dead double positive (DiO+PI+) MC38 cells was determined after analysis using a FACSCalibur with CellQuest 3.3 software (Becton Dickinson). In order to determine the percentage of CD107a+ cells, restimulated spleen cells were incubated for 2 h with MC38 cells in the presence of monoclonal anti-CD107a antibody conjugated with APC (BioLegend). Afterwards, cells were stained with anti-CD45 V500, anti-CD4 FITC, anti-CD8 PE-Cy7 and anti-CD49b PE and analyzed using a FACS Fortessa with Diva software (Becton Dickinson).
7
Extracellular recordings using multi-electrode probes
8
Extracellular recordings using multi-electrode probes
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We performed extracellular recordings using multi-electrode silicon probes (A1×32-Edge-5mm-20–177-A32, NeuroNexus) with 32 channels spaced by 20 μm. The recording electrodes were controlled with micromanipulators (Luigs&Neumann) and coated with DiO lipophilic dyes (Life Technologies) for post hoc identification of the electrode track. We recorded the bandpass-filtered (0.1 Hz to 7.5 kHz) signals at 30 kHz using an Intan system (RHD2000 USB Interface Board, Intan Technologies).
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